Disrupting circadian control of peripheral myogenic reactivity mitigates cardiac injury following myocardial infarction

Abstract Aims Circadian rhythms orchestrate important functions in the cardiovascular system: the contribution of microvascular rhythms to cardiovascular disease progression/severity is unknown. This study hypothesized that (i) myogenic reactivity in skeletal muscle resistance arteries is rhythmic and (ii) disrupting this rhythmicity would alter cardiac injury post-myocardial infarction (MI). Methods and results Cremaster skeletal muscle resistance arteries were isolated and assessed using standard pressure myography. Circadian rhythmicity was globally disrupted with the ClockΔ19/Δ19 mutation or discretely through smooth muscle cell-specific Bmal1 deletion (Sm-Bmal1 KO). Cardiac structure and function were determined by echocardiographic, hemodynamic and histological assessments. Myogenic reactivity in cremaster muscle resistance arteries is rhythmic. This rhythm is putatively mediated by the circadian modulation of a mechanosensitive signalosome incorporating tumour necrosis factor and casein kinase 1. Following left anterior descending coronary artery ligation, myogenic responsiveness is locked at the circadian maximum, although circadian molecular clock gene expression cycles normally. Disrupting the molecular clock abolishes myogenic rhythmicity: myogenic tone is suspended at the circadian minimum and is no longer augmented by MI. The reduced myogenic tone in ClockΔ19/Δ19 mice and Sm-Bmal1 KO mice associates with reduced total peripheral resistance (TPR), improved cardiac function and reduced infarct expansion post-MI. Conclusions Augmented microvascular constriction aggravates cardiac injury post-MI. Following MI, skeletal muscle resistance artery myogenic reactivity increases specifically within the rest phase, when TPR would normally decline. Disrupting the circadian clock interrupts the MI-induced augmentation in myogenic reactivity: therapeutics targeting the molecular clock, therefore, may be useful for improving MI outcomes.

For Panels C-E, * denotes P<0.05 for ZT7 versus ZT19 at a given transmural pressure or phenylephrine concentration.

Cre-WT
A.   Myogenic tone was assessed in cremaster arteries isolated from naïve wild-type mice at ZT7 (white circles). The arteries were then incubated with 1µmol/L CKI-7 for 30 minutes and myogenic tone was re-assessed (grey circles). * denotes p<0.05 (paired t-test comparisons). Naïve wild-type mice were treated with a single dose of PF670462 (50mg/kg i.p. within 2 hours of ZT7); cremaster arteries were harvested at ZT7 the following day. For comparison purposes, the 30mg/kg PF670462 is reproduced from Figure 2I of the main manuscript. Compared to the 30mg/kg dosage, 50mg/kg PF670462 has no appreciable effect. Western blot assessment of ERK1/2 phosphorylation in naïve wild-type cremaster skeletal muscle resistance arteries maintained at 40 mmHg (n=10) or 5 minutes following a 40 mmHg to 100 mmHg pressure step (n=10). A representative western blot image is displayed above. A total of 10 independent experiments were performed, each with 1 vessel sample from each group. After pooling, the data were normalized to the mean of the 40 mmHg group. To account for differences in western blot conditions, the data are analyzed with a paired t-test. * denotes p<0.05. Western blot assessment of ERK1/2 phosphorylation in naïve wild-type cremaster skeletal muscle resistance arteries pre-treated with 10 µmol/L PF670462 (30 minutes) and then subjected to a pressure step from 40 mmHg to100 mmHg (lysates prepared 5 minutes post-pressure step; n=10). A representative western blot image is displayed above. A total of 10 independent experiments were performed, each with 1 vessel sample from each group. To account for differences in western blot conditions, the data are analyzed with paired statistical test (Wilcoxon test    Shown are the uncropped, annotated western blots probing wild-type cremaster skeletal muscle artery lysates for total ERK1/2 (p42/p44) and phosphorylated ERK1/2. The artery lysates were prepared at Zeitgeber times 3, 7, 11, 15, 19 and 23 following a pressure step from 40 mmHg to 100 mmHg (5 minutes duration). All blots contain a standard (S1) to ensure similar detection; blots 4-5 possess a diluted standard (S2). Data from these blots are incorporated into Figures 2M and 2N; data from Blot 4 are displayed as a cropped image in Figure 2M (sample lanes 3-8; all data shown, excluding standards).

Supplemental Figure 16: Uncropped western blot images -ERK phosphorylation (wild-type).
Shown are the uncropped, annotated western blots probing wild-type cremaster skeletal muscle artery lysates for total ERK1/2 (p42/p44) and phosphorylated ERK1/2. The artery lysates were prepared at Zeitgeber times (ZT) 7 and 19, either without a pressure step (transmural pressure 40 mmHg; TMP: 40) or five minutes following a pressure step from 40 mmHg to 100 mmHg (TMP: 100). In certain experiments, arteries were pretreated with 10 µmol/L PF670462 (30 minutes) prior to the pressure step (denoted with a "PF" beside the Zeitgeber time). All blots contain a standard to ensure similar detection (S1 and S2; S2 is diluted in relation to S1). Data from these blots are incorporated into Figure 2N and Supplemental Figures 8-9; data from Blot 6 are displayed as a cropped image in Figure 2N

Supplemental Figure 17: Uncropped western blot images -ERK phosphorylation (TNF KO).
Shown are the uncropped, annotated western blots probing cremaster skeletal muscle artery lysates for total ERK1/2 (p42/p44) and phosphorylated ERK1/2. The artery lysates were prepared from tumor necrosis factor knockout mice (TNF KO) at Zeitgeber times (ZT) 7 and 19 five minutes following a pressure step from 40 mmHg to 100 mmHg (TMP: 100). All blots contain a standard to ensure similar detection (S1 and/or S2; S2 is diluted in relation to S1). Data from these blots are incorporated into Figure 2O; data from Blot 2 are displayed as a cropped image in Figure 2O (sample lanes 1 and 2).      Supplemental Table 8: JTK_CYCLE analysis of gene expression in cremaster arteries isolated from naïve wild-type and tumor necrosis factor knockout mice.
Data are means ± standard error measurements, where n equals the number of samples (each sample generated from 1 mouse). JTK P is the Bonferroni-adjusted minimal P value calculated by JTK_CYCLE. Acrophase is the Zeitgeber time of the sinusoidal rhythm peak calculated by JTK_CYCLE. * denotes a statistically significant circadian rhythm (P<0.05).
Abbreviations: CK1 = casein kinase 1; TNF = tumor necrosis factor; TNFR = tumor necrosis factor receptor; TNF KO = tumor necrosis factor knockout.  Supplemental Table 9: JTK_CYCLE analysis of clock gene expression in arteries isolated from mice with myocardial infarction or sham surgical procedure.
Data are means ± standard error measurements, where n equals the number of samples (each sample generated from 1 mouse). JTK P is the Bonferroni-adjusted minimal P value calculated by JTK_CYCLE. Acrophase is the Zeitgeber time of the sinusoidal rhythm peak calculated by JTK_CYCLE. * denotes a statistically significant circadian rhythm (P<0.05). Abbreviation: MI = myocardial infarction.

Figure Condition
Gene n n n n n n JTK P Period Acrophase Amplitude